Destruction or Reconstruction: A Subtle Liaison between the Proteolytic and Signaling Role of Protein Ubiquitination in Spermatogenesis.

Ubiquitination is one of the most diverse forms of protein post-translational modification that changes the function of the landscape of substrate proteins in response to stimuli, without the need for "de novo" protein synthesis. Ubiquitination is involved in almost all aspects of eukaryotic cell biology, from the best-studied role in promoting the removal of faulty or unnecessary proteins by the way of the ubiquitin proteasome system and autophagy-lysosome pathway to the recruitment of proteins in specific non-proteolytic signaling pathways, as emerged by the more recent discoveries about the protein signature with peculiar types of ubiquitin chains. Spermatogenesis, on its own, is a complex cellular developmental process in which mitosis, meiosis, and cell differentiation coexist so to result in the continuous formation of haploid spermatozoa. Successful spermatogenesis is thus at the same time a mixed result of the precise expression and correct intracellular destination of structural proteins and enzymes, from one hand, and the fine removal by targeted degradation of unfolded or damaged proteins as well as of obsolete, outlived proteins, from the other hand. In this minireview, I will focus on the importance of the ubiquitin system all over the spermatogenic process, discussing both proteolytic and non-proteolytic functions of protein ubiquitination. Alterations in the ubiquitin system have been in fact implicated in pathologies leading to male infertility. Notwithstanding several aspects of the multifaceted world of the ubiquitin system have been clarified, the physiological meaning of the so-called ubiquitin code remains still partially elusive. The studies reviewed in this chapter provide information that could aid the investigators to pursue new promising discoveries in the understanding of human and animal reproductive potential.

Towards defining an 'origin'-The case for the mammalian acrosome.

The acrosome is an organelle unique to sperm cells, thought to be indispensable for fertilization. In globozoospermia the main defect is characterized by the absent or severely malformed acrosome. Males with severe globozoospermia, to have the ability to father their own children, are committed to one of the most advanced and expensive method of assisted conception: in vitro fertilization with intracytoplasmic sperm injection (ICSI). However, rates of successful ICSI fertilization remain still very poor because globozoospermic spermatozoa have not the ability to trigger oocyte activation. It is emerging that the acrosome is not only fundamental for the release of lytic content to penetrate oocyte's coats; the strategies of oocyte penetration and activation by mammalian spermatozoa uncover less conventional and unexplored roles for acrosomal components. Discovered as cellular organelle in the early 1920s [1], the nature of the acrosome and its biogenesis are still matter of debate. In this review, I discuss the distinct classifications proposed in the years for the acrosome, providing also a glance on the intimate relationship that exists between acrosomal constituents and function. Emphasis is particularly devoted to the recent work on the acrosome biogenesis.

Dynamic of VE-cadherin-mediated spermatid-Sertoli cell contacts in the mouse seminiferous epithelium.

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid-Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.

Dynamic of contribution of UBPy-sorted cargo to acrosome biogenesis: effects of its derailment in a mouse model of globozoospermia, the infertile Vps54 (L967Q) mutant.

The sperm acrosome is a specialized vacuole, a member of the family of cell-specific lysosome-related organelles. Its exocytosis, the acrosome reaction, is a crucial event during fertilization. The released acrosomal contents promote sperm penetration through the investments of the oocyte, whereas the membranous components of the acrosome are involved in sperm-oocyte interaction/fusion and oocyte activation. The way that these functionally distinct acrosomal costituents reach the vacuole during its biogenesis remains poorly understood. The biosynthetic pathway and a consistent supply from the endosomal system have recently been documented. We use immunogold electron microscopy to determine the contribution of endosome cargo-sorting during step-by-step mouse acrosomogenesis. The chosen proteins of this study were UBPy (ESCRT-DUB), together with endosome compartment markers EEA1 and pallidin. The latter is described here for the first time in male germ cells. This new insight expands our knowledge of acrosomogenesis, confirming the plasticity of the endosomal system in supporting cell-type-specific functions. We also study wobbler mice, whose Vps54 mutation causes motor neuron degeneration and male infertility. Use of electron/immunoelectron microscopy and immunofluorescence enabled us to establish that the lack of an acrosome in wobbler spermatozoa is attributable to an early block in acrosome biogenesis and that the mislocalization of acrosome-destined proteins, potentially involved in the signaling events leading to oocyte activation, is possibly responsible for wobbler infertility, even after intracytoplasmic sperm injection.

The deubiquitinating enzyme UBPy/USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells.

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPy(C748A) caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

USP8/UBPy-regulated sorting and the development of sperm acrosome: the recruitment of MET.

The acrosome is a peculiar vacuole that at fertilization undergoes the acrosome reaction (AR), an event unique in the sperm life. Contents released promote sperm penetration through oocyte's investments; membranous components are involved in sperm-egg interaction/fusion. Therefore, both constituents play a role in fertilization. The biogenesis of this vacuole, however, has not been clarified yet; recently, it has been proposed as a novel lysosome-related organelle (LRO). Our research focuses on the involvement of the endosomal pathway in acrosomogenesis starting from the early phases. The trafficking sorted by USP8/UBPy, an endosomal regulator recently described as a compelling candidate for male fertility gene, was investigated in comparison to that of SP56, a marker of the biosynthetic pathway. Mouse spermatids were double/triple immunolabeled and examined by confocal microscopy. The contribution of the vesicular traffic assisted by the cortical microtubule array was also evaluated in nocodazole-treated spermatids. USP8/UBPy-sorted cargo contributes early to acrosomogenesis and its trafficking is microtubule mediated. It was identified, through co-immunoprecipitation/co-immunolocalization assays, that the membrane receptor MET, described herein for the first time in spermatids, as an USP8/UBPy-target substrate is delivered to the acrosome. MET and USP8/UBPy still colocalize in epididymal spermatozoa. Following the AR, MET and USP8/UBPy show a distinct fate. MET, in particular, translocates at the PAS, the post acrosomal segment known to harbor sperm-borne factors involved in oocyte activation. Overall, our results support the concept of the acrosome as a LRO and provide evidence for the identification of MET as a tyrosine kinase receptor that may play a role in fertilization.

The ESCRT-deubiquitinating enzyme USP8 in the cervical spinal cord of wild-type and Vps54-recessive (wobbler) mutant mice.

Usp8 is a deubiquitinating enzyme that works as regulator of endosomal trafficking and is involved in cell proliferation. "In vivo" USP8 is predominantly expressed in the central nervous system and testis, two organs with highly polarized cells. Considering that neuronal cell functionality is strictly dependent on vesicular traffic and ubiquitin-mediated sorting of the endocytosed cargo, it could be of relevance to investigate about USP8 in neuronal cells, in particular motor neurons. In this study, we found that USP8 is expressed in the gray and white matter of the spinal cord, labeling neuronal cell bodies, axonal microtubules and synaptic terminals. The glia component is essentially USP8-immunonegative. The partial colocalization of USP8 with EEA1 in motor neurons indicates that USP8 is involved in early endosomal trafficking while that with Vps54 suggests an involvement in the retrograde traffic. The variant Vps54(L967Q) is responsible for the wobbler phenotype, a disorder characterized by motor neuron degeneration. We searched for USP8/Vps54 in wobbler spinal cord. The most worth-mention result was that wobbler oligodendrocytes, in contrast to the wild-type, are heavily USP8-immunoreactive; no significant modification was appreciated about the cellular expression of mutated Vps54. On the other hand, as to the neuronal intracellular localization, both USP8 and Vps54(L967Q) did not show the typical spot-like distribution, but seemed to accumulate in proteinaceous aggregates. Collectively, our study suggests that in neuronal cells USP8 could be involved in endosomal trafficking, retrograde transport and synaptic plasticity. In disorders leading to neurodegeneration USP8 is upregulated and could influence the neuron-oligodendrocyte interactions.

Characterization of the constitutive pig ovary heat shock chaperone machinery and its response to acute thermal stress or to seasonal variations.

Reduced oocyte competence causes the lower fertility reported in domestic sows during the warm months of the year. Somatic cells express heat shock proteins (HSPs) to protect themselves from damage caused by thermal stress. HSPs are classified as molecular chaperones and control the correct folding of newly synthesized or damaged proteins. The present work performed a comprehensive survey of the different components of the heat shock chaperone machinery in the pig ovary, which included the HSP40, HSP70, HSP90, and HSP110 families, as well as heat shock factors (HSF) 1 and 2. Pig ovarian follicles constitutively expressed different members of these families; therefore, we examined their ability to respond to heat stress. In order to take into account the role of the complex follicular architecture, whole pig ovaries were exposed to 41.5°C for 1 h. This exposure significantly disrupted oocyte maturation and determined the upregulation of the HSP70, HSP40, HSPH1, HSPA4, HSPA4L, HSF1, and HFS2 genes, whereas expression levels of HSP90A and HSP90B, as well as those of genes unrelated to heat stress were not altered. Unexpectedly HSP and HSF expression levels changed only in oocytes but not in cumulus cells. Cumulus-oocyte complexes isolated from ovaries collected in summer showed the same pattern as those collected in winter. We conclude that the HSP chaperone machinery is constitutively fully operational in the pig ovary. However, following thermal stimuli or seasonal variations, cumulus cell HS-related gene expression remains unchanged, and only oocytes activate a response, suggesting why this mechanism is insufficient to preserve their competence both in vitro and in vivo.

RalGPS2 Interacts with Akt and PDK1 Promoting Tunneling Nanotubes Formation in Bladder Cancer and Kidney Cells Microenvironment.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell-cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.

USP8, a regulator of endosomal sorting, is involved in mouse acrosome biogenesis through interaction with the spermatid ESCRT-0 complex and microtubules.

Ubiquitin-specific peptidase 8 (USP8) is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously, we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle that is indispensable for fertilization, the acrosome; USP8 might be involved in vivo in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and trafficking/transport (MIT) domain, we verified whether USP8 effectively associates with spermatid microtubules by microtubule cosedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 (endosomal-sorting complex required for transport-0) and microtubule structures; USP8/ESCRT-0-labeled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 can directly link, via its MIT domain, the labeled vesicles/developing acrosome to microtubules, which could favor both acrosome assembly and shaping. VPS54, the vacuolar-sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosome-related organelle.

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant.

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.

cAMP-Epac2-mediated activation of Rap1 in developing male germ cells: RA-RhoGAP as a possible direct down-stream effector.

Rap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase-activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis.

Expression of the deubiquitinating enzyme mUBPy in the mouse brain.

Mouse UBPy (mUBPy) is an ubiquitin-specific protease which belongs to a family of deubiquitinating enzymes (DUBs) implicated in several cellular processes related to both cell growth and differentiation. Previously, Northern blot analysis revealed an important expression of mUBPy in the testis and brain. However, a more comprehensive map of mUBPy localization in the central nervous system (CNS) is still lacking. In this study, we mapped the distribution of mUBPy in the mouse brain using nonradioactive in situ hybridization and immunohistochemical techniques. In general, transcript and protein showed a similar and widespread distribution. In particular, mUBPy was strongly expressed in the hippocampal formation, septal region, ventral pallidum, preoptic nucleus, periventricular nucleus of hypothalamus, compact part of the substantia nigra, ventral tegmental area, cochlear nucleus and granular cell layer of cerebellum. A moderate expression of mUBPy was found in the amygdaloid complex, supraoptic nucleus, arcuate and ventromedial nuclei of hypothalamus, lateral hypothalamic area and lateral and reticular part of the substantia nigra. Double labelling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is generally expressed in neurons and, in specific regions, also in oligodendrocytes. Moreover, by using antisera to TH and mUBPy we found that mUBPy is localized in dopaminergic neurons. The different distribution of mUBPy in the distinct regions of the brain suggests that it could be related to different deubiquitinating processes; in particular, in the areas where it is expressed at high levels, mUBPy could exert a specialized function through its interaction with specific protein substrates.

Signaling events during male germ cell differentiation: update, 2006.

The intracellular transduction of exogenous and cell-autonomous stimuli triggers the transformation of a multipotent stem cell, the spermatogonion, into a highly differentiated, motile and fertile cell, the spermatozoon. This differentiation process is mediated by cell-cell contact and via key players including hormones, growth factors, and cytokines. Female hormones, estrogens and progestins, play a role in the production and functionality of spermatozoon. New findings, however, reconsider the direct action for estrogens on male germ cells while progestins work through non-canonical receptors. Similarly, testosterone, the male hormone, besides acting through its receptor expressed in the somatic cells of testis, seems to work by means of non-classical mechanisms. The recent identification of growth factors, transcriptional regulators, and media for in vitro growth of spermatogonial stem cells should now make it feasible to unravel the entire spermatogenic process. A peculiar feature of the meiotic cycle is the maintenance of condensed chromatin so that DNA duplication is prevented and reduction of genome is achieved. Recently, molecular mechanisms that lead to such a condensation have been discovered. Junctional intercellular complexes between Sertoli and germ cells are critical for coordinating spermatogenesis. Molecular players involved in such cell-cell communication have been identified in Sertoli cells. Now, there is also a need for unravelling the germ cell molecules involved. These issues are the major topics which are discussed here with the goal to suggest a possible answer.

The dynamic of the apical ectoplasmic specialization between spermatids and Sertoli cells: the case of the small GTPase Rap1.

Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception.

Acrosome biogenesis: Revisiting old questions to yield new insights.

The acrosome is a unique membranous organelle located over the anterior part of the sperm nucleus that is highly conserved throughout evolution. This acidic vacuole contains a number of hydrolytic enzymes that, when secreted, help the sperm penetrate the egg's coats. Although acrosome biogenesis is an important aspect of spermiogenesis, the molecular mechanism(s) that regulates this event remains unknown. Active trafficking from the Golgi apparatus is involved in acrosome formation, but experimental evidence indicates that trafficking of vesicles out of the Golgi also occurs during acrosomogenesis. Unfortunately, this second aspect of acrosome biogenesis remains poorly studied. In this article, we briefly discuss how the biosynthetic and endocytic pathways, assisted by a network of microtubules, tethering factors, motor proteins and small GTPases, relate and connect to give rise to the sperm-specific vacuole, with a particular emphasis placed on the endosomal compartment. It is hoped that this information will be useful to engage more studies on acrosome biogenesis by focusing attention towards suggested directions.

Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant.

The acrosome is a unique organelle that plays an important role at fertilization and during sperm morphogenesis and that is absent in globozoospermia, an inherited infertility syndrome in humans. At the light of recent experimental evidence, the acrosome is considered a lysosome-related organelle to whose biogenesis both the endocytic and biosynthetic pathways contribute. Vps54 is a vesicular sorting protein involved in the retrograde traffic; the recessive Vps54(L967Q) mutation in the mouse results in the wobbler phenotype, characterized by motor-neuron degeneration and male infertility. Here we have investigated the spatio-temporal occurrence/progression of the wobbler fertility disorder starting from mice at post-natal day 35, the day of the first event of spermiation. We show that the pathogenesis of wobbler infertility originates at the first spermiogenetic wave, affecting acrosome formation and sperm head elongation. Vps54(L967Q)-labeled vesicles, on the contrary of the wild-type Vps54-labeled ones, are not able to coalesce into a larger vesicle that develops, flattens and shapes to give rise to the acrosome. Evidence that it is the malfunctioning of the endocytic traffic to hamper the development of the acrosome comes out from the study on UBPy. UBPy, a deubiquitinating enzyme, is a marker of acrosome biogenesis from the endocytic pathway. In wobbler spermatids UBPy-positive endosomes remain single, scattered vesicles that do not contribute to acrosome formation. As secondary defect of wobbler spermiogenesis, spermatid mitochondria are misorted; moreover, with the progression of the age/disease also Sertoli-germ cell adhesions are compromised suggesting a derailment in the endocytic route that underlies their restructuring.

The PH-PxxP domain of RalGPS2 promotes PC12 cells differentiation acting as a dominant negative for RalA GTPase activation.

RalGPS2 is a guanine nucleotide exchange factor for RalA GTPase characterized by a C-terminal Pleckstrin Homology (PH) domain; this GEF is endogenously expressed in PC12 cells and in rat brain but its role in PC12 cells and in cell differentiation is actually unknown. Here we have shown that transient expression of RalGPS2-PH-PxxP domain in PC12 and PC12-TrkA cells induces high level of neurite outgrowth; this differentiation is comparable with that of PC12 cells treated with RalGPS2 siRNA. Stable PC12 cell lines expressing the PH-PxxP domain of RalGPS2 have been generated; in these cell lines the PH-PxxP domain acts as a dominant negative for RalA activation, promotes cells differentiation and re-directs NGF signals towards MAPKs. Furthermore it has been also demonstrated that the PH-PxxP domain of RalGPS2 induces microspikes formation a typical feature of cells in which the Cdc42 GTPase is constitutively activated.

Expression pattern of mUBPy in the brain and sensory organs of mouse during embryonic development.

Mouse UBPy (mUBPy) belongs to the family of ubiquitin-specific processing proteases (UBPs). In this study we have investigated the expression of mUBPy in the brain and sensory organs of mouse at different embryonic stages (E9, E11, E13, E15, E17, E19) and during the postnatal stages P0, P1, P2, P4 and P5 using Western blot and immunohistochemistry. mUBPy-immunoreactive cell bodies first appeared at stage E11 in several brain regions, particularly in the walls surrounding the vesicles and the ventricles. Subsequently, at stage E13, new mUBPy-positive cells appeared in the corpus striatum, the caudate nucleus, the thalamus, the epithalamus, the hypothalamus and the pons. At E15 the mUBPy pattern was very similar to that observed at E13, whereas at stage E17 mUBPy-immunoreactivity significantly decreased and a high number of mUBPy-immunoreactive cells was found only to line the third ventricle and within the mantle layer of the fourth ventricle. At E19 and P0, no mUBPy-immunoreactive element was found in the brain. At the postnatal stages P2 and P5, mUBPy-positive cells were detected in all subdivisions of the brain, with high concentrations in several cortex regions. Double labeling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is expressed both in neurons and astrocytes. Outside the brain, mUBPy was detected, from stage E11, in the eye, within the lens and the cornea, in the inner ear, at the level of the cochlear and vestibular systems and in the olfactory epithelium. The spatio-temporal expression of mUBPy suggests that the enzyme may be involved in neuroregulatory processes during embryogenesis.